Most medicines/pharmaceuticals are administered systemically, for example orally, intravenously, by vaccine, intramuscularly or the like. Notable exceptions are stents coated with active ingredients, certain respiratory formulations delivered directly to the lungs, certain radiotherapies which are directed to target areas and certain dermatological, ophthalmological, and otological treatments which are administered topically.
Nevertheless, when appropriate, it would be advantageous to be able to deliver the pharmaceutical primarily to a diseased tissue or organ, because this would reduce the dose required and also minimize side effects. Such an approach would be particularly advantageous for two main areas of medicine: a) the administration of growth factors and cytokines capable of activating the growth and differentiation of resident stem cells in a particular tissue. Because of the potent biological activity of these molecules, it would be desirable to limit their action to the intended tissue, with minimal or no spillover to the rest of the body; b) the delivery of cancer chemotherapeutic agents because if the cancerous tissue could be targeted specifically then it may allow the administration of higher doses to the targeted cells while minimizing the terrible toxic side effects of the same, at least to a significant extent.
In more acute situations such as in heart attacks and strokes better treatments may be possible, particularly those directed to regenerate the damaged tissue, if the organs affected could be specifically targeted. In chronic situations, such as Parkinson disease, diabetes, or pulmonary fibrosis, local administration of agents capable to reconstitute the deficient cell type(s) have the potential to improve the prognosis of the disease.
However, reproducible delivery of active ingredients to target tissue or a target organ in a therapeutically effective manner is influenced to a large extent on the components (including excipients) employed, their physical characteristics, the dose and the mode of delivery.
The present disclosure provides a pharmaceutical formulation for parenteral, especially intra-arterial, administration to a target tissue comprising particles containing an active ingredient and a biodegradable polymer excipient, wherein 30% or more of the particles have a diameter of 25 microns or less and the formulation is substantially free of particles with a diameter greater than 50 microns, such that where the formulation is administered upstream of the target tissue the ability of the active ingredient to pass through the target tissue and pass into systemic circulation is restricted. That is to say the active ingredient is retained in the target tissue while its ability to pass through the target tissue and pass into systemic circulation is severely restricted or abolished. Thus, in a particular aspect of the invention a pharmaceutical formulation for parenteral administration to a cardiac tissue is provided, said pharmaceutical composition comprising particles containing an active ingredient and a biodegradable excipient, wherein 90% or more of the particles have a diameter of between 10 and 20 microns and the formulation is substantially free of particles with a diameter greater than 50 microns and less than 5 microns, such that where the formulation is administered upstream of the target tissue the ability of the active to pass through the target tissue and pass into systemic circulation is restricted. In one embodiment at least 90%, of the particles of the pharmaceutical invention have a diameter that is between 15 and 20 microns.
In an aspect of the invention a pharmaceutical formulation for parenteral, e.g. intra-arterial, administration to a cardiac tissue is provided, said pharmaceutical composition comprising particles containing an active ingredient, selected from the group consisting of HGF and IGF-1, and a biodegradable excipient, wherein 90% or more of the particles have a diameter of between 10 and 20 microns and the formulation is substantially free of particles with a diameter greater than 50 microns and less than 5 microns, such that where the formulation is administered upstream of the cardiac tissue the ability of the active to pass through the cardiac tissue and pass into systemic circulation is restricted.
Whilst not wishing to be bound by theory it is thought that formulations of the present disclosure, when administered in the arterial blood upstream of the target tissue or organ, are carried into the target tissue or organ by the circulation and due to the particle size and distribution lodge, in other words are trapped or caught in the capillaries in the tissue or organ, which are about 5-10 μm in diameter. Particles lodging in capillaries and blocking blood flow is not generally desirable but the number of capillaries affected by the formulation of the disclosure is relatively small, particularly as the formulation enables very low therapeutic doses to be employed. Furthermore, the biodegradable excipient melts, dissolves, degrades or in some way disassociates itself from the active and thus ultimately the “blockage” is removed. Thus the movement of the particle is restricted/retarded by lodging in capillaries, a reversible process which returns the capillaries back to the natural condition after a short period. Retarding the movement of the particle for a short period allows the active to be maintained in the vicinity of the target for an appropriate amount of time to facilitate local action or absorption of the active into the extravascular space of the tissue.
The formulation is designed such that most, if not all the active is released from the particle while immobilized in the target tissue vascular bed. Once the active load is released the particle is designed to be degraded and its constituent materials released into the general circulation to be either metabolized or eliminated through the liver and/or kidney.
The present disclosure provides a pharmaceutical formulation for parenteral administration to a target tissues comprising particles containing an active ingredient and a biodegradable excipient, wherein 30% or more of the particles have a diameter of 25 microns or less and the formulation is substantially free of particles with a diameter greater than 50 microns, such that where the formulation is administered upstream of the target tissue the active is retained in the target tissue or organ for a therapeutically effective period.
In particular the formulations of the present disclosure allow lower quantities of active ingredients to be employed because the majority of active is retained in the target tissue rather than being taken into the systemic circulation. This seems to increase the therapeutic window of the active. That is to say the dose range over which the ingredient is therapeutically active is increased allowing smaller absolute quantities to be administered. Local administration of a lower dose means that side effects are likely to be minimised.
Suitable doses are, for example in the range 0.05 μg/Kg to about 10 μg/Kg, such as 0.1 μg/Kg to about 0.5 μg/Kg, in particular 0.15, 0.2, 0.25, 0.35, 0.4 or 0.45 μg/Kg.
Administrating lower doses locally for therapeutic effect is particularly important for potent molecules, for example growth factors, which are known to have potential to stimulate oncogenesis. These potentially harmful side effects limit the utility of such molecules even though in the right circumstance they produce therapeutically beneficial effects.
The formulations of the present disclosure do not employ microspheres comprising a polystyrene, silica or other non-biodegradable bead with active ingredient attached thereto, because enduring resilient materials i.e. non-biodegradable materials such as polystyrene and silica may cause damage to local capillaries, and may act as foreign bodies and produce local inflammatory reactions. Moreover, such non-biodegradable beads might eventually gain access to the systemic circulation and may then, for example accumulate in distant tissue such as the lungs and liver, all of which are undesirable.
Generally, each particle will comprise active and excipient. It is not intended that the description of the formulation refer to discrete particles of active and separate particles of biodegradable polymer in simple admixture.
Substantially free of particles over 50 microns as employed supra is intended to refer to formulations that meet the criteria to be administered as a parenteral formulation set down in the US pharmacopeia and/or European pharmacopeia.
In one embodiment substantially free may include containing less than 5% of said particles, particularly less than 1%, for example less than 0.5%, such as less than 0.1%.
In one embodiment the at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% such as at least 99% of the particles have a diameter of 25 microns or less.
In one embodiment the particle size is in the range 6 to 25 microns, such as 10 to 20 microns, particularly 15 or 20 microns, for example at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% such as at least 99% of the particles are the relevant size or within said range. Thus in one embodiment of the invention at least 95%, at least 98% or at least 99% of the particles of the pharmaceutical composition have a diameter of between 10 and 20 microns. In another embodiment at least 95%, at least 98% or at least 99% of the particles of the pharmaceutical composition have a diameter of between 15 and 20 microns.
In one embodiment the formulation does not contain particles less than 1 micron in diameter.
In one embodiment the formulation does not contain particles less than 5 microns in diameter.
In one embodiment at least 30% of the particles with the active are retained in the target tissue after administration, for example at least 40%, at least 50%, at least 60%, at least 70%, such as at least 80% or more of the active particles are retained.
In one embodiment the active particle is retained in the target tissue or organ for a period in the range 5 minutes to 24 hours, for example 30 minutes to 5 hours, such as 1, 2, 3 or 4 hours.
The period that the formulation is retained in the relevant tissue or organ depends primarily on the excipient or the combination of excipients employed. Thus the properties required from the excipient in vivo are that:                it is biocompatible (i.e. generally non-toxic and suitable for administration to humans and/or animals),        within an appropriate time frame after administration it contributes to maintaining the particle integrity sufficiently for the particle movement to be retarded by, for example lodging in a capillary or arteriole in the target tissue or organ, and        it is biodegradable (that is to say it is capable of being processed or metabolised) by the body to release the active and after the active has been released.        
Thus a biodegradable polymer excipient suitable for use in the present disclosure is a polymer or co-polymer that does not have a long residency time in vivo, ie would not include entities such a polystyrene, polypropylene, high density polyethene and material with similar properties. Biodegradable polymers must be non-toxic and broken down into non-toxic sub-units preferably locally, such that the amount of circulating fragments/debris from the excipient are minimised.
Suitable excipients can be found in the United States Pharmacopeia (USP) and include inorganic as well organic, natural and man-made polymers. Examples may include polymers such as polylactic acid, polygycolide or a combination of the same namely polylactic co-glycolic acid, polycaprolactone (which has a slower rate of biodegradation than polylactic co-glycolic acid), polyhydroxybutyrate or combinations thereof. Polyurethanes, polysaccharides, proteins and polyaminoacids, carbohydrates, kitosane, heparin, polyhyaluronic acid, etc may also be suitableThe excipient is generally in the form of a particle, an approximate sphere (microsphere) to which the active can be attached or with which the active is associated or incorporated within.
Liposomes are not biodegradable polymer excipients within the meaning of the present disclosure. Liposomes are vesicles of a phospholipid bilayer generally comprising cholesterol. For diseases such as myocardical infarction induced by arterio sclerosis cholesterol levels are monitored as one of the risk factors for the disease and thus it may be advisable to avoid administering cholesterol containing formulations to such patients. In addition patients with liver cirrhosis may have increased difficulty metabolising lipids and dietary fats, therefore administration of liposomes to such patients may not be advisable.
In one embodiment the biodegradable excipient is not a hydrogel (a continuous phase of a corresponding colloidal dispersed phase).
Thus, both the rate of “release” of the active and the rate of “dissolution” of the particle can be altered by altering the excipient or/and the method of binding the active to the excipient, so for example employing polycaprolactone would provide a particle which takes longer to dissolve or disintegrate than a corresponding particle employing polylactic co-glycolic acid. If the active is embedded within the excipient it will be released more slowly than if it is on the surface of the particle. If on the surface and bound by electrostatic charge it will be released faster than if covalently bound.
In one embodiment the excipient comprises polylactic co-glycolic acid.
In one embodiment substantially all the particles, for example 80, 85, 90, 95, 96, 97, 98, 99 or 100% of the particles comprise polylactic co-glycolic acid.
In one embodiment the polylactic co-glycolic acid is in the ratio 75:25 respectively.
In one embodiment the excipient comprises two or more distinct polymers, the term polymer includes co-polymers.
In one embodiment the excipient may include an acrylate polymer, for example a methacrylate polymer.
In one embodiment the particle comprises alginate.
In one embodiment the excipient comprises a biodegradable form of polyurethane.
In one embodiment the excipient is in the form of a microsphere.
In one embodiment the disclosure employs a polyvinyl alcohol microsphere formulation.
In one embodiment the microspheres are not albumin.
In one embodiment the active(s) employed are encapsulated within a biodegradable coating for example selected from the Eudragit range.
In one embodiment one or more active molecules are embedded within the particle.
For the active compounds to perform, as described in the present disclosure, they need to be administered into the circulation as a microparticle which because of its size, morphology and composition will travel with the blood flow to reach its target tissue. At the target, the particle should release its active load in a controllable manner. To accomplish this goal, once unloaded, the particle should be degraded and its constituents either metabolized or delivered into the systemic circulation to be eliminated by the normal excretion systems of the body.
To accomplish these goals the microparticles should fulfill the following characteristics:
The microparticles should be of uniform size and morphology in order to insure that they reach and become lodged at the designed level of the circulatory system. Uniformity of size and shape is better controlled when the particles are spherical.
Most capillary beds allow free passage of particles with a diameter of <6 microns in diameter, the microspheres of this disclosure should have a diameter >6 microns, and preferably of ˜15 microns. Particles in the range of 20 microns in diameter or larger lodge into pre-capillary arterioles or arterioles and block the blood flow to several capillaries at once. Therefore, they might create microscopic infarctions. Thus for the delivery of regenerative therapies the most suitable diameter of the microspheres is in the range of 15 microns. In addition, however, particles having diameters of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 are contemplated for use according to the present invention.
The time required to release the active compound once they have reached their target could range from minutes to days and even weeks, depending on the type of microsphere and the therapeutic goal.
The microspheres should be made with a biodegradable and non-toxic compound. The stability of the particle and its degradation time will depend on the composition and type of the microsphere. It might be designed to deliver its load before it starts degrading; alternatively it might be designed so that the delivery of its load occurs as the particle disintegrates.
The nature of the polymer used as excipient, its size, lability of the bonds between the monomers and degree of cross-linking, if any, will affect the rate of release of the active as well as the stability and degradability of the particle.
In all embodiments, the microspheres should be stable enough in solution for them not to substantially break or degrade during their administration into the circulation and the time required for them to reach the target vascular bed.
In a suitable embodiment of the disclosure, each particle will carry a single type of active compound. When a mixture of compounds is thought to be beneficial for therapeutic purposes, a mixture of microparticles, each loaded with a single type of compound, may be administered. This design simplifies the production of the therapeutic compounds and offers greater therapeutic flexibility, thereby allowing individualized medicaments to be prepared rapidly to meet the patient's individual specific needs.
In one embodiment a particle(s) employed has/have only one type of active molecule bound to it/them.
In one embodiment a particle(s) employed has a mixture, such as two, three or four active molecules bound to it.
The active compound might be loaded onto the particle at the time of its formation and, for example be dispersed throughout the particle.
The active compound may be encapsulated inside the particle where the excipient forms the shell of the microsphere.
In one embodiment active(s) are bound to a particle(s) by covalent bonds, for example a polypeptide or protein is bonded to a microsphere through cross-linking by treatment with an aldehyde such as formaldehyde or glutaldehyde, for example by emulsifying the microsphere (or ingredient of the microspheres) in the presence of the active(s), a suitable aldehyde and homogenizing the mixture under conditions suitable for forming particles of the required size. Alternatively the active may be bonded to a carboxylate group located on the excipient microsphere.
In one embodiment the active(s) are bound to a particle(s) by electrostatic forces (charge).
In one embodiment the active(s) are bound to a particle(s) through a polyelectrolyte such as, for example comprising sodium, potassium, magnesium and or calcium ions with chloride counter ions in aqueous solution.
In one embodiment the active(s) are bound to a particle(s) between layers of polyelectrolytes.
The active compound may be loaded on the surface of the particle either by charge (electrostatic forces) or covalently bound. In one embodiment the active(s) is/are bound to the particle by electrostatic charge.
In one embodiment the active(s) is/are bound to the particle by polyelectolytes, for example by means of a polyelectrolyte shell covering the particle onto which the active attaches by charge.
The active compound may form a single layer on the surface of the particle or might be deposited in multiple layers either contiguous or separated by polyelectrolyte layers.
The active compound may be bound to the particle by means of “linkers” which on one hand bind to the excipient matrix and on the other to the active compound. These bonds might be either electrostatic or covalent.
The microparticles may for example be stabilized by lyophilization. Microparticle may also be stable when frozen.
In one embodiment the excipient is degraded rapidly in the range of minutes to hours, or over a longer period such as weeks to months.
In one embodiment the formulation is such that once in the circulation one or more actives is/are rapidly released for example in period in the range of 1 to 30 minutes to about 1 to 12 hours.
In one embodiment the disclosure relates to a mixed population of particles that is to say, particles with different rates of “dissolution”, which may be used to provide a formulation with controlled or pulsed release.
Thus formulations of the disclosure can comprise particles with different release kinetics and degradation rates.
In one embodiment the active is released over a period of 1 to 24 hours.
In one embodiment the active is released over a period of 1 day to 7 days.
Thus in one or more embodiments all the formulation of the disclosure is metabolized within 7 days of administration.
In one embodiment once in the circulation of the individual, the active(s) is/are released very slowly, over a period weeks to months, for example 1 week to 1, 2, or 3 months.
In one embodiment the population of particles is well characterized and for example has the same characteristics. That is to say the physical and/or chemical properties of each particle fall with a narrow defined range.
In one embodiment the size of the microspheres is monodispersed.
Thus in one embodiment the particles of the formulation have mean particle size with a small standard deviation, for example at least 68% of particles have a size+/−1 micron of the mean, such as 99% of particles have a particle size+/−1 micron of the mean (eg 15+/−1 microns). In addition, compositions wherein the particles have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% of particles within +/−1 micron of the mean are contemplated by the present invention.
In one embodiment the formulation comprises a population of particles characterized in that the populations contains at least two distinct types of particle, for example the distinct particles may have different actives, coatings, particle size or a combination of the same.
In one embodiment the disclosure relates to a mixed population of particles comprising particles of active in admixture with particles of one or more further distinct actives.
It appears the particle size and distribution of the formulation influences the in vivo profile of the formulation including how the formulation in distributed in the tissue. It seems that is insufficient to simply have a mean particle size within the range 10 to 20 microns because this allows some particles to have a much larger particle size and also a much smaller particle size. This variation can cause problems in vivo because, for example the small particles are not retained with the relevant tissue and the larger particles can damage the tissue.
The amount of active:excipient employed may be in the ratio 1%:99% w/w, 5%:95% w/w, 10%:90% w/w, 20%:80% w/w, 30%:70% w/w, 40%:60% w/w, 50%:50% w/w, 60%:40% w/w, 70%:30% w/w, 80%:20% w/w or 90%:10% w/w, depending on what release profile is required. If the active is required to be release quickly or immediately in vivo a higher ratio of active to excipient may be chosen.
In one embodiment the microsphere employed has a half life of about 16 hours.
In one embodiment the formulation is lyophilized.
In another embodiment the formulation is frozen.
The particles of the disclosure are not magnetic to an appreciable extent.
The active ingredient may be any medicine or pharmaceutical that may be administered in the form of a particle according to the disclosure.
In one embodiment 15×106 particles (microspheres) are administered, such as 14×106, 13×106, 12×106, 11×106, 10×106, 9×106, 8×106, 7×106, 6×106, 5×106, 4×106, 3×106, 2×106 or 1×106 particles are administered.
A particle as employed herein may comprise, for example micronized drug, semi-solid or hydrated entities such as proteins or biologically derived actives formulated as discrete particles provided the particle maintains its structure for a sufficient period to perform the required function. The disclosure also extends to particles with a liquid core provided that the external integrity of the particle is such that is can perform its function in vivo. The disclosure does not extend to particles with a gas core.
Microspheres may be fabricated by emulsifying a polymer solution, followed by evaporation of solvent. In other instances monomers are emulsified followed by thermal or UV polymerization. Alternatively, a polymer melt is emulsified and successively cooled to solidify the droplets. A size reduction of the emulsion can be obtained by homogenizing or sonicating the bulk. The microspheres can be collected by filtering and/or centrifuging the reaction mixture.
Biodegradable microspheres and microcapsules of biopolymers for the controlled release and targeted delivery of different pharmaceutical compounds and therapeutic macromolecules have been long known in a number of forms, particularly those of relatively large diameters as described in the present disclosure (see D. D. Lewis “Biodegradable polymers and drug delivery systems” M. Chasin and R. Langer, editors (Marcel Dekker, New York, 1990); J. P. McGee et al., J. Control. Release 34:77, 1995).
Microspheres and microcapsules are routinely produced by mechanical-physical methods such as spraying constituent monomers into microdroplets of the size followed by either a drying or polymerization step. Such microparticles can also be formed through emulsification followed by removal of the emulsifying solvent (B. Miksa et al., Colloid Polym. Sci. 273: 47, 1995; G. Crotts et al., J. Control. Release 35:91, 1995). The main challenge of these methods is the production of a monodisperse population of particles in shape and size. This, for example can be achieved employing a technique of flow focusing in which a capillary nebulizer is used to form microdroplets of the proper size. In the process the components are submerged into a harvesting solution/solvent which serves to dissolve/suspend the microparticle components, followed by evaporation of the solvent to provide solidified microparticles.
This process may require that all the components of the microparticle be combined into a single mixture (the focused compound) from which are generated the microdroplets that will form the microparticles. As many of the polymers used for drug delivery are hydrophobic while most therapeutic macromolecules, and particularly proteins, are hydrophilic the mixture requires emulsifying to ensure a homogeneous composition is obtained before the microparticles are formed.
Alternatively particles may be prepared, for example by aspirating a solution of active into microspheres in a convection current, from a nozzle with a net electric charge toward a plate or entity with a counter charge, in an anode/cathode type arrangement.
In one embodiment particles employed have a net electric charge, for example a positive charge or negative charge. This may, for example assist the particle's movement being retarded in the target tissue or organ. This net charge may be balanced in the formulation for administration by counter ion spheres (for example without active) of a small dimension, for example less than 5 micron, which are not retained within the target tissue after administration.
In one embodiment the active ingredient is a biological molecule or derived therefrom, for example a protein such as an antibody or a growth factor, a cytokine or combination of entities.
In particular the formulations of the disclosure are, particularly useful for targeting/activating resident stems cells found in the relevant tissue.
In one preferred embodiment the disclosure is used to activate the resident stems, progenitors and/or precursors of a particular tissue or organ to stimulate regeneration of said tissue or organ.
In one embodiment the disclosure relates to localized administration of ligands for the receptors expressed by the stem cells present in the post-natal tissue for initiation of regeneration of the same. The ligand may, for example be a growth hormone as described herein.
In one embodiment the ligands are administered to activate the receptors present on the most undifferentiated stem cells present in each target tissue. These cells express the so-called “multipotency genes”, such as Oct 4, Sox2, Nanog, etc. and they have a potent regenerative capacity (hereafter known as Oct4-expressing stem cells).
In one embodiment the ligand is administered to the heart to minimize and/or regenerate tissue damage for example caused by myocardial infraction.
When an artery is obstructed the main effect is a loss of the tissue downstream from the obstruction. The specific consequence of the obstruction of a coronary artery is a myocardial infarction (MI) which results in the irreversible loss of a portion of the cardiac muscle. This loss results in a diminution of the contractile capacity of the myocardium and the pumping capacity of the heart which, when significant enough, limits its capacity to provide the appropriate cardiac output and produces a serious and progressive limitation of the person's capacity (reviewed in Nadal-Ginard et al., Circ. Res. 2003; 92:139).
In the USA and the EU alone over 1.5 million MIs are treated every year and there are over 11 million MI survivors (American Heart Association, 2007; British Heart Association, 2007). Of these, over 30% die during the first year post-infarct. The survival post-MI depends in large measure on the size of the infarct (% of muscle mass lost) due to the ischemic event. When the loss affects ˜40-45% of the left ventricular mass it produces an irreversible cardiogenic shock which is uniformly lethal (Page et al., 1971. N. Engl. J. Med. 285; 133). This segmental myocardial loss produces a reorganization of the reminder myocardium with increased cell death by apoptosis, hypertrophy of the surviving myocytes, increased fibrosis of the tissue and dilation of the ventricular chamber (Pfeffer, M. A. & Braunwald, E., 1990. Circulation 81:1161). This reorganization, known as “remodeling”, because of its negative effects on contractility, frequently evolves into cardiac failure (CF). After the first episode of CF post-MI the average survival is <5 years with a yearly mortality of ˜18% (American Heart Association, 2000).
Most or all the therapies to treat the loss of parenchymal tissue, due to ischemia or to other causes are directed to preserve or improve the function of the surviving tissue. In the case of an MI, all the therapies presently in use to treat the consequences of loss of cardiac contractile muscle are directed to preserve or enhance the contractile function of the surviving tissue and to reduce the continued loss of these muscle cells by apoptosis or by necrosis (see Anversa & Nadal-Ginard, 2002. Nature 415:240; Nadal-Ginard et al. 2003. Circ. Res. 92:139). At present there is not a single approved therapy designed to regenerate or to replace the myocytes lost in the MI and, in this manner, restore the contractile function of the heart. Moreover, all the experimental approaches described until now are directed to improve the blood flow to the ischemic/necrotic area by stimulating the increase in the capillary network, most often by directly or indirectly delivering to the affected area growth factors such as vascular endothelial growth factor (VEGF) either in protein form or in the form of cDNA. Not a single therapy is directed to the resident stem cells in the tissue to stimulate them to multiply and differentiate in order to regenerate together the parenchyma and microcirculation lost by the vascular accident.
The goal of the therapeutic approaches to the acute MI is to restore the blood flow the damaged muscle as soon as possible to prevent further muscle loss. These reperfusion therapies include the use of thrombolytic agents, balloon angioplasty or bypass surgery. In the USA in 1998 >500,000 angioplasties and a similar number of surgical bypasses were performed. These therapies often are successful in restoring blood flow to the ischemic muscle, but none are able to replace a single muscle cell already lost at the time of the intervention. If this loss has been substantial, the long term consequence is an inability to generate the required cardiac output which will inexorably evolve to terminal heart failure.
Until now the only option to effectively treat terminal heart failure has been cardiac transplant with all the medical (immunosuppressive therapy), logistic and economic problems that it entails. Even if these problems could be circumvented, the shortage of donors makes this therapy available to >1% of the patients in cardiac failure.
The formulations of the present disclosure allow the administration of the therapeutically active molecules to be administered in a form where the tissue or organ such as the heart can be targeted specifically to regenerate tissue, for example damaged by obstruction of an artery, by stimulating stem cells already present in the tissue to regenerate.
Stem Cell Therapy for Tissue Regeneration.
Recently some experimental approaches have been developed as alternatives to organ transplantation which are targeted to replace some of the cells lost by the organ or tissue of interest. These procedures have been modeled in the success of the bone marrow transplants carried out for over half a century. The capacity of a small population of cells in the bone marrow to generate all blood cell types, when transplanted in an immunologically competent individual, proved convincingly that adult tissues contained “stem cells” capable to generate and regenerated a tissue or a whole organ. This conceptual breakthrough has led to the developments of experimental approaches to repair damaged tissues using different types of stem cells isolated from the individual to be treated (autologous cell therapy) or isolated from an individual different from the one to receive them (heterologous cell therapy). These cells are either isolated on mass or first expanded in culture before being transplanted to produce the desired repair of the affected tissue. These cell therapy approaches take advantage of the natural regenerative properties of the stem cells for tissue regeneration.
The term “stem cell” is used here to identify a cell that has the properties of self-renewal (generate more cells like itself), is clonogenic (can be expanded starting from a single cell) and it is pluripotent; that is it can produce a progeny which will differentiate into different cell types, often present in the tissue where they reside. That is, the cells originated from a stem cell will acquire particular cellular specializations characteristic of the tissue or organ from which the stem cell originated or into which it is transplanted (Stem Cells: A Primer. 2000. National Institutes of Health USA).
The term “pluripotent” refers to cells which are capable of differentiating into a number of different cell types. In the context of this application the term “tretrapotent” refers to a cell that although it might not be totipotent (capable of generating a whole individual), it is capable to generate four different cell types; e.g. cardiomyocytes, vascular endothelial and smooth muscle cells and connective tissue fibroblasts.
The term “progenitor cell” refers to a descendant of a stem cell which has already committed to a particular differentiation pathway and, therefore, has a more restricted differentiation potential than the stem cell. The progenitor cell has a great capacity of amplification and, although it does not yet express markers of differentiation, it has the capacity to create a progeny that is more differentiated than itself. For example, the term may refer to an undifferentiated cell or to a cell that has differentiated to an extent short of its final differentiation. This cell is capable of proliferation and giving rise to more progenitor cells, therefore having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells. In particular, the term progenitor cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues. A progenitor cell is more differentiated than a true stem cell because it has already restricted somewhat the multipotency of the stem cell from which it originated.
As used herein unless the context indicates to the contrary stem cell refers to stem cells, progenitor cells and/or precursor cells.
Cellular differentiation is a complex process typically occurring through many cell divisions. A differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on. While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors as has been recently demonstrated with the iESCs (induced embryonic stem cells) (Takahashi et al., 2007. Cell 131:1-12).
A “precursor cell” is a descendant of the progenitor cell which has gone further down the differentiation pathway and has become committed to differentiate into a single cell type even though it might not yet express any of the identifiable markers for this cell type. The precursor cell is usually the one undergoing the last round of amplification before the appearance of the identifiable differentiated phenotype.
Stem cells are present in the inner cell mass of the blastocyst, the genital ridges of the early embryo, the placenta and in the majority of tissues of the adult animals, including the human. In contrast to the stem cell derived from the inner cell mass of the blastocyst, in general, the stem cells isolated from adult tissues are a mixture of true stem cells, progenitors and precursors together with cells at the earliest stage of their final differentiation. Adult stem cells have now been identified in practically all tissues originated from each of the three embryonic cell layers (endoderm, mesoderm and ectoderm), ranging from the bone marrow, central and peripheral nervous system, all connective tissues, skin, gut, liver, heart, inner ear, etc.
It appears that these adult stems cells have regenerative capacity. Surprisingly, despite the high prevalence, severity and high economic costs of the ischemic cardiopathy in all developed countries, until recently there has been no search for procedures targeted to the regeneration of the adult myocardium. One of the reasons for this anomaly has been that until very recently the heart was considered a terminally differentiated organ without any intrinsic regenerative capacity of its contractile cells (MacLellan, W. R. & Schneider, M. D. 2000. Annu Rev. Physiol. 62:289; Reinlib. L. and Field, L. 2000. Circulation 101:182; Pasumarthi, K. R. S, and Field, L. J. 2002. Circ. Res. 90:1044; MacLellan, W. R. 2001. J. Mol. Cell. Cardiol. 34:87; Perin, E. C. et al 2003. Ciculation 107:935; see Anversa, P. and Nadal-Ginard, B. 2002. Nature 415:240; Nadal-Ginard, B. et al 2003 Circ. Res. 92:139). This concept was based on the experimentally well documented fact that in the adult heart the vast majority of cardiomyocytes are terminally differentiated and their capacity to re-enter the cell cycle has been irreversibly blocked. Thus, there is no doubt that these myocytes are not able to reproduce to generate new myocytes.
One consequence of the prevailing concept of the myocardium as a tissue without regenerative potential has been that all the so-called experimental “regenerative therapies” implemented until now have been based on the introduction within the damaged heart of different cell types that either are fetal myocytes or are believed to have some potential to differentiate into this cell type or into capillaries and microarterioles in order to substitute for the cells lost during the infarct. In this manner animal experiments have been performed transplanting fetal and adult skeletal muscle precursor cells, fetal cardiac myocytes, and embryonic stem cells either in their undifferentiated state or after their commitment to the cardiomyocyte pathway (Kocher et al., 2001. Nature Med. 7: 430).
With the exception of the skeletal muscle precursor (which are incapable of converting to cardiocytes and are unable to become electrically coupled to the myocardial cells) (Menasche et al., 2001. Lancet 357: 279; C Guo et al. 2007. J Thoracic and Cardiovasc Surgery 134:1332) which can be autologous, all other cell types listed are by necessity of heterologous origin and, therefore, have either to be accompanied by immunosuppressive therapy or the transplant is rapidly eliminated by the immune system. The fact is that none of these approaches have proved to be very effective in preclinical assays and all have many pitfalls.
One of the most intriguing characteristics of some of the adult stem cells is their “plasticity”. This property refers to the fact that when certain stem cells are placed within a tissue different from the one they originated from, they can adapt to this new environment and differentiate into the cell types characteristic of the host tissue instead of the donor tissue. Although the extent and nature of this plasticity for many cell types still remains controversial (Wagers & Weissman, 2004. Cell 116:636-648; Balsam et al., 2004 Nature 428, 668-673; Murray et al., 2004. Nature 428, 664-668; Chien, 2004. Nature 428, 607-608), it has spawned countless preclinical protocols and clinical trials.
Among the adult stem cells described until now, those from the bone marrow have been the most studied and those that have shown a greater degree of “plasticity” (Kocher et al., 2001. Nature Med. 7: 430). Also widely used have been the so-called “mesenchymal stem cells” derived from adipose tissue (Rangappa, S. et al 2003. Ann. Thorac Surg 75:775).
The capacity of bone marrow and adipose-tissue derived stem cells to re-populate damaged areas of different tissues and organs, the relative ease of their isolation, together with the earlier work of Asahara et al (1999; Circ. Res. 85: 221-228), has proven advantageous for the objectives of cell therapy to regenerate to cardiac muscle in experimental animals (Orlic et al., 2001. Nature 410:701; Orlic et al., 2001. Proc. Natl. Acad. Sci. USA 98:10344; Nadal-Ginard et al., 2003. Circ. Res. 92:139) and in the human (Tse et al., 2003. Lancet 361:47; Perin et al., 2003. Circulation 107:2294). Although it has been questioned by some, (Balsam, L. B. et al. 2004. Nature 428: 668; Murry, C. E. et al. 2004. Nature 428: 664), it is clear that bone marrow derived stem cells under certain conditions are capable to generated cardiomyocytes, capillaries and microarterioles, particularly when transplanted in the border area of an experimental myocardial infarction. (Quaini, F., et al., 2002. New Engl. J. Med. 346:5; Bayes-Geis, A. et al., 2003. Cardiovasc. Res. 56:404; Bayes-Genis, A. et al., 2004. Eur. J. Heart Fail. 6:399; Thiele, H. et al., 2004. Transplantation 77:1902). No similar information is available from the numerous clinical trials of cell therapy with either bone marrow- or adipose tissue-derived stem cells because no reliable histopathological data is available for evaluation.
A major drawback of the techniques used for myocardial cell therapy is the complexity and inefficiency of the cell transplantation procedure itself. When the cells are transplanted through the coronary arterial tree, only 3-5% remains in the myocardium while the rest is spread throughout the body. If the cells are injected directly into the myocardium, it requires either a thoracotomy or the use of complex and time consuming instrumentation (Noga-type systems) in order to identify the target area. This technique requires specialized operators and it is only available in specialized medical centers. In addition, the intramyocardial injections, either by transendocardial (Noga) or transepicardial (surgical) route still delivers <50% of the cells to the tissue.
Without exception, all cell therapy approaches used up to the present time to produce myocardial regeneration post-myocardial infarction either in experimental animals or in the human have been developed completely ignoring the fact that the myocardium has an intrinsic regenerative capacity represented by its resident stem cells (Nadal-Ginard, B., at al., 2003. J. Clin. Invest. 111:1457; Beltrami et al., 2003. Cell 114:763-776; Torella, D., et al., 2004. Circ. Res. 94:514; Mendez-Ferrer, S. et al., 2006. Nature Clin. Prac. Cardiovasc. Med. 3 Suppl 1:S83; Torella et al., 2007. Cell. Mol. Life. Sci. 64:661).
As indicated above, until recently the accepted paradigm considered the adult mammalian heart as a post-mitotic organ without regenerative capacity. Although over the past few years this concept has started to evolve, all the experimental and clinical approaches to myocardial regeneration have continued to be based on the old dogma. For this reason all cardiac regeneration protocols have been based on cell transplantation in order to provide the myocardium with cells with regenerative potential.
It now seems that when formulations of the present disclosure are administered under appropriate conditions that the intrinsic regenerative capacity of the “stem cells” resident in the tissue or organ (such as the heart) can be stimulated or activated to regenerate the tissue or organ.
Thus in one aspect the disclosure provides a method for the regeneration of solid tissues in living mammals, including humans, which include the local delivery of ligands for the receptors expressed by the stem cells present in the post-natal tissue to be regenerated. These are cells that when stimulated physiologically or pharmacologically multiply in situ and differentiate into the parenchymal cells characteristics of the tissue or organ that harbors them.
New cardiomyocyte formation has been detected in both the normal heart and in pathological conditions such as MI and cardiac failure (Beltrami, A. P. et al., 2001. New Engl. J. Med. 344:1750; Urbanek, K. et al., 2003. Proc. Netl. Acad. Sci. USA. 100:10440; Nadal-Ginard, B. et al., 2003. J. Clin. Invest. 111:1457; Nadal-Ginard, B. et al., 2003. Circ. Res. 92:139). Interestingly, these new myocytes are significantly more abundant at the border zone of MIs where they are an order of magnitude more abundant than in the myocardium of age matched healthy individuals. These observations suggested that the adult human myocardium has the capacity to respond to acute and chronic increases in cell death with an abortive regenerative process that attempts to replace the dead myocytes (Anversa, P. & Nadal-Ginard, B. 2002. Nature 415: 240; Anvrsa, P. and Nadal-Ginard, B. 2002. New Engl. J. Med. 346:1410; Nadal-Ginard, B. et al., 2003. Circ. Res. 92:139).
Adult cardiac stem cells (CSCs) were first described in 2003 (Beltrami et al. 2003. Cell 114:763-776) and confirmed by several authors in the same and other species (see Torella, D., et al., 2004. Circ. Res. 94:514; Mendez-Ferrer, S. et al., 2006. Nature Clin. Prac. Cardiovasc. Med. 3 Suppl 1:S83; Torella et al., 2007. Cell. Mol. Life. Sci. 64:661). These CSCs are self-renewing, clonogenic and multipotent because they give rise to cardiomyocytes, endothelial and smooth muscle vascular cells as well as to connective tissue fibroblasts. They were identified by expression of membrane markers associated with stem cells such as c-kit, the receptor for SCF, Sca I, MDR-1 and Isl-I. It is now clear that the new myocytes formed in the adult heart are derived from the CSCs resident in the myocardium. These CSCs, when injected at the border of an infarct, have the capacity to regenerate the contractile cells and the microvasculature lost as a consequence of a massive MI (Beltrami, et al., 2003. Cell: 114:763-776; Laugwitz, et al. 2005; Mendez-Ferrer et al., Torella et al., 2006; Torella et al., 2007).
In the heart of a healthy individual, almost all CSCs are in a resting state (G0) or cycling very slowly during the lifespan of the organism. At any given time, only a very small fraction of these cells is active, undergoing replication and differentiation just enough to replace the cells that die by wear and tear. In contrast, a large fraction of the CSCs—sometimes the majority—is activated in response to a physiological or pathological stress. In general, there is a direct correlation between the magnitude of the stress and the number of CSCs that became activated in response. This number of activated CSCs is in turn also directly correlated to the number of new myocardial cells generated. This response, which occurs from mouse to human (Nadal-Ginard, B. et al., 2003. Circ. Res. 92:139), reveals the existence of a biochemical pathway triggered by the stress that results in the activation of the CSCs.
The communication between the resident stem cells and their environment, at least in the myocardium, is regulated by a feed-back loop between the cardiomyocytes, that sense the changes in wall stress produced by increased physiological or pathological demands in cardiac output, and the stem cells responsible to produce an increase in muscle mass through the generation of new contractile cells and microcirculation to nurture them. The myocytes have a stereotypical response to stress independently of whether it is physiological or pathological (Ellison et al., 2007. J. Biol. Chem. 282: 11397-11409). This response consists in rapidly activating expression and secretion of a large battery of growth factors and cytokines such as HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor 1), PDGF-β (platelet-derived growth factor β), a family of FGFs (fibroblast growth factor), SDF-1 (stromal cell-derived factor 1), VEGF (vascular endothelial growth factor), erythropoietin (EPO), epidermal growth factor (EGF), activin A and TGF β (transforming growth factor β), WINT3A and neurogeulin among others. This secretory response, in addition to stimulate the hypertrophy of the myocytes themselves through an auto/paracrine loop, also triggers the activation of CSCs in their vicinity because these cells express receptors for these myocyte-secreted factors and respond to them. This response activates genetic pathways downstream of the receptor that are responsible for cell survival, multiplication and differentiation. In addition, the activation of these receptors also activate a feed-back loop in the CSCs themselves which stimulates the production of the respective ligand by the CSCs, thus putting in place a self-sustained response which, in response to a single stimulus, can remain active for several weeks or until the increased mass produced has restored the myocardial wall stress to normal levels. Therefore, the CSCs respond to a paracrine stimulus with an auto/paracrine response which allows the maintenance of a sustained response to a short lived stimulation. Thus, normal cardiac cellular homeostasis is maintained through a continuous feed-back between myocytes and CSCs to produce and maintain the appropriate contractile muscle mass required to generated the needed blood cardiac output. The myocytes, which are unable to divide, depend on the CSCs to maintain or increase their cell number and the capillary density to guaranty their oxygen and nutrient supply. The CSCs, on the other hand, depend and respond to the biochemical cues produced by their surrounding myocytes to regulate their resting vs activated state.
In addition to the tissue-specific stem cells described above, we have recently found that the myocardium of mammals, including the human, as well as most other tissues, contain a small population of very undifferentiated cells that have many similarities to the embryonic stem cells (ESCs) which have been known for a long time to be multipotent; that is, a single cell is capable, when placed in the proper environment, to generate a whole organism identical to the one from which it originated. The main characteristic of these cells is their expression of a battery of so-called “multipotency genes” such as Oct4, Sox2, Nanog, etc (see U.S. provisional application Ser. No. 61/127,067) that confer multipotency to these cells, so that, independently of their tissue of origin they seem capable to give rise to most, if not all cell types of the body. In particular, Oct4-expressing cells isolated from the adult heart are capable to give raise to skeletal muscle, neurons, heart, liver, etc. Their regenerative capacity seems more robust and broader than that of the tissue-specific stem cells.
We believe that the Oct4-expressing cells are the origin of most, if not all, the tissue-specific stem cells of every organ and that their stimulation is the main source of the regenerative capacity of every individual tissue. Therefore, the stimulation of these cells is a primary target for the therapeutic approaches described herein.
Independently of their ability and/or efficiency to generate myocardial cells, when a large number of stem cells are introduced into a tissue, regardless of their tissue of origin, they have an important paracrine effect when transplanted into the myocardium and other tissues, as has been proven experimentally. The complex mixture of growth factors and cytokines produced by the transplanted cells have a potent anti-apoptotic effect over the cardiomyocytes and other cells in the area at risk and also in the activation of the endogenous stem cells that multiply and differentiate into muscle cells and microvasculature. This paracrine effect starts very soon after the cell transplantation and can be documented in vitro.
It seems from the work performed in the examples herein that to stimulate the resident stem cells of a tissue (including the Oct4 expressing cells), in this case the myocardium, the growth factors and cytokines produced by the stressed myocytes and to which the CSCs respond could be as or more effective than cell transplantation to trigger a regenerative response. A combination of insulin-like growth factor 1 and hepatocyte growth factor may be particularly effective.
In one embodiment resident stems cell are activated, for example to stimulate regeneration of the tissue, to increase muscle density and/or cell function of target cells.
If the target cells are cardiac muscle then the increased function would, for example be greater/increase contractile function.
If the target cells are kidney cells, in a renal failure kidney patient, then the increased function may be increased capacity to generate EPO.
If the target cells are pancreatic cells then the increased function may be increased capacity to generate insulin.
It seems that formulations of the disclosure are able to stimulate/activate stems cells resident in “mature tissue” thereby obviating the need to administer “stem-cell” therapy to the patient as the resident stems are stimulated to undergo mitosis and grow.
Stimulating resident stems cells is distinct from angiogenesis. Angiogenesis is the process of stimulating growth of capillaries (which may be in tissue or tumors) (see Husnain, K. H. et al. 2004. J. Mol. Med. 82:539; Folkman, J., and D'Amore, P. A. 1996. Cell 87:1153). In contrast, when formulations of the present disclosure employing appropriate ligands are administered a stem cells resident in the tissue, such as pluripotent cells, progenitor cells and/or a precursor cells are activated to generate new/additional tissue cells such as muscle cells.
All the regenerative approaches described until now have severe limitations either because of the nature of their biological target, the regenerative agent used and/or the route and mode of administration. The vast majority of so-called regenerative therapies have been directed to regenerate the capillary network of the ischemic myocardium using a variety of biological factors, such as vascular endothelial growth factor (VEGF), whose main role is to stimulate the growth of the surviving endothelial cells in the damaged tissue in order to expand the capillary network and improve the blood supply (Isner, J. M. and Losordo, D. W. 1999. Nature Medicine 5:491; Yamaguchi, J., et al., 2003. Circulation 107:1322; Henry, T. D., et al., 2003. Circulation 2003. 107:1359). These therapies neither attempt nor accomplish the regeneration of the parenchymal cells that perform the characteristic function of the tissue or organ; e.g. contractile cardiomyocytes in the heart, hepatocytes in the liver, insulin-producing β cells in the pancreas, etc. At best, these therapies have had modest effects and none of them has become part of standard medical practice. On the other hand, all the regenerative therapies designed to replace the functional cells of the tissue or organ have until now been based in the transplantation of cells believed to be able to take on the characteristics of the missing cells in the target tissue. These approaches are still in clinical trials. A main drawback for all the regenerative approaches used has been to deliver the regenerative agent to the damaged tissue and limit their spread throughout the rest of the body. This is a serious problem even when the regenerative agents are administered through the coronary arterial tree of the tissue to the treated. In the cases of myocardial cell therapy by coronary administration, only a very small fraction of the cells administered is retained in the heart, while the majority (>95%) rapidly enters the systemic circulation and it is distributed throughout the body. This also occurs when the regenerative agents are directly injected into the myocardium either trans-epicardially or trans-endocardially, as has been repeatedly demonstrated with the administration of a cell suspension. In addition, the trans-epicardial administration requires exposing the heart through a thoracotomy, while the trans-endocardial administration requires a sophisticated, time consuming and expensive procedure to map the endocardium to identify the regions suitable for injection (a Noga-type instrument), a procedure available in a very limited number of centers and the participation of an expert manipulator. In both cases, at best 50% of the administered compound is retained in the damaged are while the remainder is spread either throughout the thoracic cavity or through the systemic circulation. The formulations of the disclosure may be used in combination with the delivery of stems cells to a target tissue or organ and increase the number that are retained locally in comparison to other delivery mechanisms.
However, this disclosure describes a novel method to regenerate the parenchymal cells (that is, the functional, “noble” cells) of a tissue or organ that is based neither on cell transplantation nor on the growth stimulation of the surviving endothelial cells in order to improve the blood supply to the tissue or organ of interest. Instead, the methods described here are based in the stimulation in situ, that is, within the tissue, of the resident stem cells of such tissue by means of local delivery of specific growth factors and/or cytokines which are able to stimulate their activation, replication and differentiation to generate the parenchymal cells lost as well as the microvasculature needed for their growth, survival and function. This is possible because most, if not all adult tissues mammalian tissues, including human tissue, contain resident stem cells which are capable, when properly stimulated, of regenerating the cell types which are specific to the tissue or organ, as well as the vascular and mesenchymal supporting cells which accompany them.
Because some of the regenerative agents that stimulate the stem cells are very active and might stimulate the growth and translocation of a variety of cells they interact with, among them latent neoplastic cells, the potential clinical application of many of these factors will require the administration of the smallest therapeutic doses in a very localized manner in order to, as much as possible, limit exposure to the cells that are to be regenerated. Thus, the more localized the administration the lower the doses required and lower the risk of undesired side effect due to stimulation of by-stander cells in the same or other organs. More specifically, the disclosure describes a new approach for the use of therapeutic doses of different growth factors administered and delivered locally, instead of systemically or tissue-wide, to produce the regeneration of specific areas of a solid tissue. Because the delivery of the active compound is localized to the damaged tissue, the therapeutic dose required is a minute fraction of what would be needed with other available delivery methods. The formulation of the disclosure is capable, among others applications, to regenerate the heart muscle and its microvasculature after a myocardial infarction and/or in chronic cardiac failure.
In one embodiment the formulation is administered at the border of the damaged tissue, for example at the border or an ischemic zone.
Suitable ligands for stems cells include growth factors such as those listed in Table 1
TABLE 1Examples of suitable stem cell ligands of the inventionHGF (hepatocyte growth factor),IGF (insulin-like growth factor) such as IGF-1,PDGF (Platelet-derived growth factor) such as PDGF-β,FGF (fibroblast growth factor) such as aFGF (FGF-1) or bFGF (FGF-2)and FGF-4,SDF-1 (stromal cell-derived factor 1),EGF (epidermal growth factor)VEGF (vascular endothelial growth factor),erythropoietin (EPO),TGF β (transforming growth factor β),G-CSF (Granulocyte-colony stimulating factor),GM-CSF (Granulocyte-macrophage colony stimulating factor),Bone morphogenetic proteins (BMPs, BMP-2, BMP-4)Activin A,IL-6,Neurotrophins for example NGF (Nerve growth factor), neuroregulin,BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3), NT-4(neurotrophin-4) and (neurotrophin-1), which is structurally unrelated toNGF, BDNF, NT-3 and NT-4TPO (Thrombopoietin)GDF-8 (Myostatin), orGDF9 (Growth differentiation factor-9).Periostin
In one embodiment the growth factor(s) employed is human.
In one embodiment the growth factor employed is selected from HGF, IGF (such as IGF-1 and/or IGF-2) and FGF, in particular HGF and IGF-1. These factors appear to be particularly effective in stimulating resident stem cells.
Combinations of growth factors may also be employed and, for example may be selected from the above-identified list, such as HGF and IGF-1 and optionally VEGF.
In one embodiment the formulation for regenerating/activating stems cells does not consist of VEGF as the only active but for example may comprise a combination of actives include VEGF.
Nevertheless the formulation is suitable for localized delivery of VEGF as angiogenesis factor.
In one embodiment the growth factor formulation is employed in combination with an angiogenesis factor, for example administered concomitantly or sequentially by the same route or a different route.
In one embodiment the formulation comprises a cytokine, for example selected from IL-1, IL-2, IL-6, IL-10, IL-17, IL-18 and/or interferon.
In one embodiment the formulation comprises combinations of actives, for example a growth factor and a cytokine.
In combination formulations then the dose of each active may, for example be the same dose employed when the active is administered alone.
The components employed in the formulations and/or methods of the disclosure, especially biological type actives may be derived from natural origin.
In one embodiment a biological type active employed is prepared by recombinant DNA technology.
In one embodiment the active or actives administered may be peptide fragments of a biological molecule, with the desired therapeutic effect.
In one or more embodiments the molecules employed are mutants of a biological molecule (for example a ligand of a receptor) with the desired therapeutic effect having the same, higher or lower affinity for the corresponding biological molecule.
In one embodiment the substance(s)/active employed is an aptomer (a small RNA molecule that binds to a receptor instead of the natural ligand).
In one embodiment the substance/active employed is an antibody that recognizes and binds to a target receptor, and in particular has a suitable specificity and/or avidity for the same. Desirably the antibody has the required activity to upregulate the receptor or down regulates the receptor thereby either producing activation or blocking of the same, as appropriate.
In one embodiment the active is a diaquine, which is an artificial antibody molecule that recognizes and binds to two of the receptors of interest resulting in either the activation or blocking of one and/or the other.
In one embodiment the substance/active employed is a small molecule with a molecular weight <5,000 Daltons.
In one embodiment one or more actives employed may be of synthetic origin.
For the formulation disclosed herein to target the desired organ or tissue then the formulation should be administered upstream of the organ or tissue. That is to say should be introduced into the circulation such that the flow of blood carries the formulation into the desired tissue/organ.
The formulation can be introduced upstream of an organ such as the heart employing a suitable device such as a catheter. Other major organs can be reached in this way. Similarly whilst is it rare it is also possible to use catheters to gain access to the liver.
In other instances the formulation may be introduced by strategic intra-arterial injection or by retrograde venous injection and/or cannular before the target tissue.
The formulation may also be administered by infusion or a pump driven delivery device such as a syringe pump, for example of the type employed in the administration of heparin or morphine or contrast agents during catheterization. A suitable flow rate may for example be 0.5 mL/min.
The formulation might also be administered through the so-called perfusion catheters that allow slowing down the rate of blood flow downstream from the site of the injection with an intra-arterial balloon, while maintaining perfusion of the tissue through a second lumen of the catheter.
In a particularly suitable embodiment the formulation is administered into an artery upstream of the target tissue or organ.
In one embodiment a catheter is used to deliver the formulation of the disclosure into the artery supplying the target tissue or organ. In particular, the formulation may be delivered exclusively (primarily or substantially) to the segmental artery that supplies the area of the tissue or organ.
In one embodiment the catheter employed is a balloon catheter.
In one embodiment the catheter carries a filter mesh at its distal end with a pore size sufficiently small to prevent or hinder the release of microparticle aggregates >50, 25 or 20 μm, as required.
In one embodiment the target cells are the cardiac stem cells resident in the post-natal heart.
In one embodiment the regeneration obtained includes together or separately the regeneration of cardiomyocytes and vascular structures composed of capillaries (endothelial cells) and/or arterioles (endothelium and vascular smooth muscle cells).
In one embodiment the regeneration is induced at any time after a myocardial infarction (MI) be it acute or chronic, for example 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 up to 24 hours after an acute infarction.
In one embodiment the regeneration is induced in an individual with ischemic heart disease, with or without a myocardial infarction.
In one embodiment the regeneration is induced in the hearts of individuals that have developed cardiac failure (CF) either acute or chronic.
In one embodiment the regeneration is induced in individuals with ischemic, infectious, degenerative or idiopathic cardiomyopathy.
In one embodiment the target cells are the stem cells resident in the endocrine pancreas (stem cells of the islands of Langerhans).
In one embodiment the regeneration is induced in an individual with diabetes.
In one embodiment the target cells are the neural stem cells of the central nervous system (CNS).
In one embodiment the target stem cells are the neural stem cells of the spinal cord.
In one embodiment the regeneration is induced in an individual with a spinal cord lesion.
In one embodiment the target cells are the stem cells of the substantia nigra of the brain, for example in an individual with Parkinson's disease.
In one embodiment the regeneration is induced in an individual with a cerebral vascular accident (stroke).
Whilst not wishing to be bound by theory it is believed that the ligands employed in formulations of the disclosure are able to cross the blood brain barrier to treat strokes and the like. In addition, in cerebral vascular accident it is believed that the blood brain barrier becomes impaired and chemical entities can more readily pass through the barrier.
In one embodiment the target cells are the liver stem cells and for example the regeneration is induced in an individual with liver damage such as cirrhosis.
In one embodiment the target stem cells are the stem cells of the lung(s) and for example the regeneration is induced in a patient with lung damage, for example emphysema.
In one embodiment the target cells are the stem cells of the skeletal muscle and for example the regeneration is induced in an individual with a particular skeletal muscle deficit, such as osteoporosis or pagets disease.
In one embodiment the target cells are the stem cells of the epithelium.
In one embodiment the target stem cells are the stem cell of the kidneys.
Target cells as employed herein refers to the cells that are to be stimulated and which have the potential to provide the desired regeneration.
The formulation of the disclosure provides optimized parameters and materials to ensure accurate and/or reproducible dosing of the relevant active to the target tissue or organ.
In an alternative embodiment the formulations of the disclosure may be employed to treat solid tumors, by allowing local delivery of the antineoplastic to the tumor tissue, for example by intra-tumor injection.
Actives suitable for the treatment of tumors include etoposide, cyclophosphamide, genistein, cisplatin, andriamycin, vindesine, mitoguazone, fluorouracil and paclitaxil.
In one embodiment the formulation is not for the treatment of cancer.
In one embodiment the invention is not administration directly into a tumor or tissue.
The methods according to the disclosure may employ combinations of actives administered separately, for example concomitantly or sequentially, or formulated as one (one-pot) formulation.
Formulations of the disclosure may be administered as liquid solutions/suspension, for example in an isotonic carrier, for example as a buffered solution such as phosphate buffer, saline or glucose solution.
Formulations of the disclosure may optionally comprise one or more further excipients. The excipients should be suitable for administration to humans and/or animals.
In one embodiment the formulation comprises albumin in solution, which may for example stabilize the small quantities of active in the formulations, for example from 1% to 20% w/vol of albumin, such as human serum album, may be sufficient to achieve the required stabilization.
The disclosure also extends to use of as a formulation as defined herein for treatment, particularly for the treatment of myocardial infarction; ischemic heart disease; cardiac failure; ischemic, infectious, degenerative or idiopathic cardiomyopathy, sclerosis, cirrhosis, emphysema, diabetes and the like.
In one embodiment the disclosure relates to a formulation as described herein for use in treatment, particularly for treatment of an illness described above.
The disclosure also extends to methods of treatment comprising administering a therapeutically effective amount of a formulation described herein to a patient in need thereof, particularly for the treatment of a disease described above.
The disclosure also extends to use of a ligand, for example as described herein, for stimulating a resident stem cell in vivo to activate the cell.
The disclosure also includes uses of a suitable growth factor for the manufacture of a medicament for stimulate resident stem cells in vivo.
The disclosure will now be illustrated by reference to the Examples.